We therefore examined the level of both proteins in the cytoplasmic membrane of anaerobically grown E. coli by Western blot analysis (Fig. 1); FocA was exclusively membrane-associated. The results revealed reproducibly that both proteins were in the membrane fraction and that FocAStrep–C and FocAStrep–N were present at similar levels, which suggests that the FocA derivative with the C-terminal Strep-tag was marginally
less active than the N-terminally tagged protein. Nevertheless, these data suggested that both proteins were active in importing formate into the cell and the Strep-tags did not interfere with membrane insertion or transport activity. It was also noted that although FocA has a deduced molecular mass of 31 kDa, it migrated in SDS-PAGE with a mass of ∼23 kDa (Fig. 1a). This aberrant migration is characteristic of integral membrane proteins (e.g. selleck products see Ito, 1984), has been noted previously for FocA (Suppmann & Sawers, 1994), and was consistently observed with different tagged FocA preparations. Western blot analysis of membrane fractions derived from anaerobically grown MC4100 (wild type) showed a similar migration behavior to overproduced FocAStrep–N (Fig. 1b), with the exception that FocAStrep–N migrated slightly more slowly due to the additional amino acids derived from the Strep-tag. No polypeptide corresponding to this molecular weight was observed in membrane fractions derived
from REK701, which lacks FocA (Suppmann & Sawers, 1994). Taken together, these data indicate that overproduced FocAStrep–N and wild-type FocA had similar size and migration Akt inhibitor features upon SDS-PAGE analysis. Comparison of the samples of membrane fractions of MC4100 with serial dilutions of purified FocAStrep–N in Western blots allowed an estimation of the number of FocA monomers present in fermenting E. coli cells (Neidhardt & Umbarger, 1996). This equated to approximately 500 monomers of FocA. It was anticipated from earlier transcriptional studies (Sawers & Böck, 1989; Suppmann & Sawers, 1994) that FocA would not be abundant, as the focA Carnitine palmitoyltransferase II transcript is processed, thus preventing translation (Sawers,
2005b). This contrasts sharply with the amount of PflB, which, under the same conditions, constitutes nearly 3% of the cytoplasmic protein (roughly 30 000 molecules) (Kessler & Knappe, 1996). Thus, despite the huge disparity in the cellular copy number, the coexpression of focA and pflB ensures that coordinate synthesis of both proteins is maintained. FocAStrep–N was overproduced in BL21(DE3) as described in Materials and methods and it was found to be membrane-associated. FocAStrep–N could be readily solubilized from the membrane by treatment with Triton X-100; however, the isolated protein precipitated. DDM treatment of the membrane fraction was also able to release the protein and in this case FocAStrep–N remained in the soluble fraction after ultracentrifugation. Similar results were obtained for FocAStrep–C.